Compounds and Method for Modulating Inflammatory Reactions

ABSTRACT

The present invention relates to compounds and methods for modulating, reducing or inhibiting, inflammatory reactions in a patient. Particularly, inflammatory reactions that are targeted by the present invention are cell migration, secretion of toxic products and proteolysis at a site of inflammation. Reduction of inflammation manifestations and reactions occurs by using an anti-S100 polynucleotide or polypeptide inhibitor or antagonist, which is essentially targeted against S100A8, S100A9 or S100A12, alone or in combination with other inhibitors of chemokines or immune modulating products.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/469,931filed May 21, 2009 which is a divisional of U.S. application Ser. No.10/517,319 filed Jul. 15, 2005, now U.S. Pat. No. 7,553,488, whichclaims the priority benefit of PCT/CA03/00939 filed on Jun. 20, 2003which claims priority to U.S. Provisional Application No. 60/393,520filed on Jul. 5, 2002. The contents of these applications are herebyincorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to inhibitors, antagonists and methods formodulating the factors involved in body inflammation reactions anddiseases. Particularly, the present invention relates to a method forreducing or inhibiting the symptoms and manifestations associated withbody inflammations.

BACKGROUND ART

The acute articular inflammation of gouty arthritis is caused bycrystallisation of sodium urate in an articulation. Interaction betweenmonosodium urate crystals (MSU crystals) and monocytes, platelets,synoviocytes, macrophages and neutrophils within the articulationinitiates an inflammatory response by stimulating the secretion ofproinflammatory agents and chemotactic factors from these different celltypes. Some of these mediators induce the accumulation of neutrophils,which further enhances the inflammatory response and release of oxygenradicals and proteolytic enzymes, leading to the destruction of thearticulations.

Arthritis is a chronic syndrome characterized by the inflammation ofperipheral joints, while gout manifests itself as an inflammation of thelower leg. Although the causal agents differ between the two diseases,the mechanism of migration of neutrophils is similar in both diseases.Therefore, for the sake of brevity, whenever reference hereinbelow ismade to arthritis, it should be understood as encompassing gout, sinceboth diseases are similar. There is a wide spectrum of disease severityand many patients run a course of intermittent relapses and remissionswith an overall pattern of slowly progressive joint destruction anddeformity. Persistent inflammation produces symptoms and damages tissuecausing loss of cartilage, erosion of bone matter and subluxation of thejoint. This results in a high degree of morbidity resulting in disturbeddaily life of the patient. Diagnosis of arthritis is typically carriedout by determination of rheumatoid factors in the blood and radiologicalchanges in peripheral joints.

Transendothelial migration of neutrophils is a critical stage in thedevelopment of the inflammatory reaction. To infiltrate an articulation,the neutrophils must migrate from the blood through the endothelium andthe synovial tissue. This migration occurs through a multistep process.

First, interactions between integrins, selectins and glycans mediateneutrophil rolling along the endothelium. Neutrophils are thenactivated, leading to changes in β₂ integrin to an active conformation.This change of conformation is thought to be induced by chemotacticfactors expressed by endothelial cells such as platelet activatingfactor (PAF) or interleukin-8 (IL-8). Activation of integrins causesneutrophils to adhere strongly to the endothelium, allowing them toextravasate. Once in the tissue, neutrophils follow concentrationgradients of chemoattractants such as complement peptide C5a,leukotriene B₄ (LTB₄) and IL-8.

Factors involved in neutrophil migration in gout pathogenesis remainlargely unknown. For example, while LTB₄ is known to be produced by MSUcrystal-activated neutrophils, inhibition of LTB₄ synthesis does notreduce MSU crystal-induced neutrophil recruitment in the subcutaneousair pouch model in rats. However, inhibition of PAF partially diminishesMSU crystal-induced arthritis in rabbits articulations. It has beenobserved that IL-8 can be the major cystein-x-cystein (C-X-C) chemokineinvolved in neutrophil migration in response to MSU crystals.Inactivation of IL-8 with specific blocking antibodies seems to lead toa reduction of neutrophil migration in rabbit articulations.

However, this reduction was observed 12 hours after MSU crystalsinjection, with no effect detected at earlier time points. This stronglysuggests that IL-8 is not responsible for the initiation of theinflammatory response induced by MSU crystals. However, early neutrophilmigration in response to MSU crystals is impaired in mice deficient inthe murine IL-8 receptor homologue CXCR2. Since CXCR2 does not solelybind IL-8, this suggess that other chemokines or inflammatory mediatorscould be involved at the beginning or even during the inflammatoryresponse.

Primary treatments of arthritis include first line drugs for control ofpain and inflammation classified as non-steroidal anti-inflammatorydrugs (NSAIDs), e.g., aspirin, ibuprofen, naproxen, methotrexate, etc.Secondary treatments include corticosteroids, slow acting antirheumaticdrugs (SAARDs) or disease modifying drugs (DMs), e.g., penicillinamine,cyclophosphamide, gold salts, azothipprine, levamisole, etc.

All of the above-mentioned products have a variety of toxic side effectsand most of them are cytotoxic. These drugs have limited advantages andtheir effects are mainly of short term duration. The side effects theyproduce, e.g., gastric erosion, and adverse effects on the kidneys andliver, dictate against their use over extended periods of time. Furtherthe products primarily used are costly and have low benefit-risk ratios.

There still remains a need for alternative therapies, methods, andcompositions or compounds for the modulation of inflammatory reactionswhich are moderate in cost, safe, efficient and which eliminate the needfor traditional products and their associated side effects, particularlyover prolonged daily use.

DISCLOSURE OF THE INVENTION

One object of the present invention is to provide a method for systemicmodulation of an inflammatory reaction in an individual, a human or ananimal, in needS comprising administrating to the individual aneffective amount of A chemotactic factor inhibitor, the chemotacticfactor being selected from the group consisting of an S100 protein, aprotein of the MRP family, calprotectin, and calgranulin.

The modulation can totally or partially inhibit the inflammatoryreaction or totally or partially increase the inflammatory reaction.

The inflammatory reaction may be selected from the group consisting ofarthritis, chronic polyarthritis, rheumatoid arthritis, gout, asthma,psoriasis, paraneoplastic syndrome, tumor-induced inflammatory diseases,turbid effusions, collagenosis, postinfectious arthritis, seronegativespondylarthritis, vasculitis, sarcoidosis, arthrosis, cell chemotaxis,cell migration, cell recruitement, proteolysis, oxidative burst, andradical oxydation.

The cell that can be chemoattracted by the compound and method of thepresent invention can be selected from the group consisting of aneutrophil, a monocyte, a platelet, a synoviocyte, a macrophage, alymphocyte, a leukocyte, and a phagocytic cell.

According to one object of the present invention, the administration canbe performed by intravenous, oral, intranasal, subcutaneous, topical, orintraperitoneal administration.

The method of the present invention is preferably performed on an animalthat is a mammal.

According to another object of the invention, an effective amount can bean amount of S100 protein inhibitor effective to induce inhibition oractivation of an inflammatory reaction.

An inhibitor used to performed the method according to the presentinvention can be an antibody or a fragment thereof binding to the S100protein or to a receptor or a cofactor thereof.

The inhibitor can alternatively be a sens or an anti-sens mRNA, or aninhibitor of transcription or translation of the S100 protein factor, oran inhibitor of activity acquisition of the chemokine factor.

The inhibitor can also be a peptide binding to the S100 protein.Preferably, the S100 protein targeted in the present invention is anS100A8, S100A9, or an S100A12 protein.

Another object of the present invention is to provide a composition formodulating an inflammatory reaction comprising a therapeuticallyaffective amount of a chemotactic factor inhibitor selected from thegroup consisting of an S100 protein, a protein of the MRP familly,calprotectin, calgranulin, a pharmaceutically acceptable carrier.

In accordance with the present invention there is provided the use of aS100 protein inhibitor in the manufacture of a composition formodulating inflammatory reaction.

One object of the present invention is to provide a method usinganti-S100 antibody or antagonists in the manufacture of pharmaceuticalsto reduce the manifestations and reactions of inflammation in a patientin need by an administration of the pharmaceutical for a determinedperiod of time.

Another object of the present invention is to provide a method in whichanti-S100 antibody is targeted essentially against the S100A8 and S100A9proteins.

A further object of the present invention is to provide a method,wherein an anti-S100 antibody can be used alone or in combination withone or more other antibodies, or in combination with any other immunemodulating product. The expression “immune modulating product” isintended to mean any product, compound, or agent that has an inhibitoryor stimulatory effect on at least one immunological reaction involved inany body inflammation.

Another object of the present invention is to provide a method, whereinthe anti-S100 antibody is a polyclonal or a monoclonal antibody.

Also, one object of the present invention is to provide a method inwhich a composition comprising at least one antagonist or inhibitor asdefined herein, can be in a form for subcutaneous, intravenous,intramuscular, intraarticular, oral, intranasal, or intraperitonealadministration.

Another object of the present invention is to provide a method that canbe applied to humans as well as animals.

For the purpose of the present invention the following terms are definedbelow.

The term “gout” is intended to mean a metabolic disorder related to ablood excess of uric acid, characterized by a painful articularinflammation.

The terms “modulation” or “modulating” as used herein is intended tomean reducing or increasing a reaction, such as an inflammatoryreaction. The modulation can be preferably a treatment. “Treatment” asused herein includes systemic use for the alleviation, amelioration orcontrol of inflammation, e.g. of inflammatory rheumatic or rheumatoiddisease, process, condition or event. It also includes intervention forthe alleviation, amelioration or control of the sequelae or symptoms ofinflammation, for example degeneration (e.g. of cells, epithelia ortissues), or especially swelling, exudation or effusion, or pain. Inthis context the term “treatment” is further to be understood asembracing use to reverse, restrict or control progression of anyspecified disease, process, condition, event or the like, including usefor disease modifying effect. If any of the mentioned diseases,processes, conditions or events is associated with pain, the term“treatment” preferably encompasses the alleviation, amelioration orcontrol (including temporal or permanent removal) of at least onefurther sequela or symptom in addition to pain, such as swelling,effusion, exsudation, stiffness, lack of flexibility of joints, ordegeneration, more preferably of all symptoms and most preferably of thetotal clinical picture of the respective disease, irritation ormanifestation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the MSU crystals-induced accumulation ofleukocytes in the mouse air pouch model;

FIGS. 2A to 2C illustrate the release of S100A8, S100A9, and S100A8/A9in air pouches of mice injected with MSU crystals;

FIGS. 3A and 3B illustrate that S100A8, S100A9, and S100A8/A9 stimulateneutrophil accumulation in vivo;

FIG. 4 illustrates that S100A8 and S100A9 are essentials to neutrophilaccumulation induced by MSU crystals;

FIGS. 5A and 5B illustrate the measurement of S100A8/A9 in synovialfluids and plasma of patients with gout. S100A8/A9 was measured by ELISAin (A) plasma and (B) synovial fluids of healthy donors and patientssuffering from gout, or osteoarthritis;

FIGS. 6A to 6D illustrate the neutrophil accumulation and secretion ofS100A8, S100A9, and S100A8/A9 in the air pouch exudates followinginjection of LPS;

FIGS. 7A and 7B illustrate the effect of polyclonal antibodies againstS100A8 and S100A9 on neutrophil accumulation induced by LPS;

FIG. 8 illustrates the local LPS injection inducing neutrophilia inmice;

FIGS. 9A to 9C illustrate the presence of S100A9 and S100A8/A9 in theserum of mice injected with LPS;

FIGS. 10A to 10C illustrate the accumulation of neutrophils in bloodafter i.v. injection of S100A8, S100A9, and S100A8/A9;

FIGS. 11A to 11D illustrate the mobilization of neutrophils from thebone marrow to the blood after injection of S100A8 and S100A9; and

FIG. 12 illustrates the effect of anti-S100A8 and anti-S100A9 onLPS-induced neutrophilia.

MODES OF CARRYING OUT THE INVENTION

In accordance with the present invention, there is provided a method andcompositions for the modulation of the activity of different factorsinvolved in the manifestations or reactions of body inflammation. Thefactors can cause migration of cells, such as for example but withoutlimiting it to, neutrophils, or can cause oxidation by radicals, orproteolysis by different enzymes of proteases.

The present invention shows that myeloid-related proteins (MRP) play arole in the process of neutrophil migration to inflammatory site. MRPproteins are a subfamily of S100 proteins in which three members havebeen characterized, namely S100A8, S100A9, and S100A12. These smallproteins are constitutively expressed at high levels in the cytosol ofneutrophils. S100A8 and S100A9 are also expressed by activatedendothelial cells, certain epithelial cells, keratinocytes, monocytesand activated macrophages. In the presence of calcium, S100A8 and S100A9associate noncovalently to form the heterodimer S100A8/A9.

Several proinflammatory activities have been identified for theseproteins. In vitro studies described herein bellow demonstrate thatS100A8, S100A9, and S100A8/A9 are involved in neutrophil and monocytemigration and stimulate neutrophil adhesion to fibrinogen by activatingthe β₂ integrin Mac-1. In addition, intraperitoneal injection of murineS100A8 in mice stimulates the accumulation of activated neutrophils andmacrophages. It is also shown that S100A9 and S100A8/A9 enhance monocyteadhesion to and migration through endothelial cells via Mac-1/ICAM-1interactions.

S100A8 and S100A9 play a certain role in neutrophil migration aschemotractants. The extracellular presence of S100A8/A9 has beendemonstrated in several pathologies including rheumatoid arthritis,tuberculosis and Crohn's disease. Local secretion of the proteins can bedetected in periodontal infections and during experimental murineabscesses.

One particular embodiment of the present invention is to providecompounds and a method for neutralizing the chemotractant activity ofthe S100 proteins to reduce or inhibit cell migration at a site ofinflammation.

Several observations demonstrate that S100A8 and S100A9 proteins play anessential role in the pathogenesis, for example but without limiting itto, of gout. In mice injected with MSU crystals, the proinflammatoryproteins S100A8 and S100A9, which are present in air pouch exudates,were found to induce the migration of neutrophil to the air pouch with akinetic similar to MSU crystals. In addition, inactivation of bothS100A8 and S100A9 led to a total inhibition of neutrophil accumulationin response to MSU crystals, clearly demonstrating their involvement inneutrophil recruitment in vivo. High concentrations of these MRPproteins are found in the synovial fluids of gout patients.

In another embodiment of the present invention it is shown that S100A8and S100A9 proteins are particular targets for the treatment of one ofthe most important symptoms in inflammatory patients, namely acutearthritic articular inflammation. This approach is now supported hereinbelow with the inhibition of the S100A8, S100A9 and S100A8/A9 activityby using anti-S100A8 and anti-S100A9 antibodies.

Indeed, inactivation of S100A9 by passive immunization reducesneutrophil recruitment at a low level. However, inactivation of S100A8reduces neutrophil recruitment by at least 50%. This data indicate thatS100A8 plays a more important role in MSU crystals-induced neutrophilrecruitment than S100A9.

According to another embodiment of the present invention, passiveimmunization with anti-S100A8 and anti-S100A9 prior to injection of MSUcrystals leads to a significant reduction or even total inhibition ofneutrophil recruitment at the site of inflammation.

Alternatively, injection of antibodies specific to S100 proteinsaccording to the present invention allows for the inactivation of theheterocomplex S100A8/A9, which is an important form found in the airpouch following MSU crystals injection. As S100A8/A9 is also achemotactic factor for neutrophils and induces neutrophil accumulationin vivo, it will be recognized by someone skilled in the art thatS100A8/A9 can also play a crucial role in MSU crystals-inducedrecruitment.

s anti-S100A8 and anti-S100A9 antibodies are effective to inactivate theS100A8, S100A9 and S100A8/A9 activity, and thus to prevent neutrophilrecruitment, the use of these antibodies also represents an excellentway to prevent inflammatory symptoms and reactions, such as for example,but without limiting it to, acute arthritic articular inflammation.

In another embodiment of the present invention, there are providedantibody-based therapies that involve administering antibodies specificto S100 proteins to an animal, preferably a mammal, and most preferablya human patient for treating one or more of the disclosed diseases,disorders, or conditions. Therapeutic compounds of the inventioninclude, but are not limited to, antibodies of the invention (includingfragments, analogs and derivatives thereof), peptides binding to S100proteins and nucleic acids encoding antibodies of the invention(including fragments, analogs and derivatives thereof and anti-idiotypicantibodies). The antibodies can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the inflammatory diseases, disorders, orconditions described herein. The treatment and/or prevention ofinflammatory diseases, disorders, or conditions associated withexpression and/or activity of an S100 protein inhibitor or antagonistincludes, but is not limited to, alleviating symptoms associated withthose diseases, disorders or conditions. Anti-S100 antibodies can beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding S100 polynucleotides orpolypeptides locally or systemically in the body or by directcytotoxicity of the antibody, e.g. as mediated by complement (CDC) or byeffector cells (ADCC). Some of these approaches are described in moredetail below.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines. The antibodies of the invention may be administered aloneor in combination with other types of treatments (e.g., radiationtherapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumoragents). Generally, administration of products of a species origin orspecies reactivity (in the case of antibodies) that is the same speciesas that of the patient, is preferred. Thus, in a preferred embodiment,human antibodies, fragments, derivatives, analogs, or nucleic acids, areadministered to a human or animal patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against S100 polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for therapy of disorders related to S100 polynucleotides orpolypeptides, including fragments thereof, of the present invention.Such antibodies, fragments, or regions, will preferably have an affinityfor S100 polynucleotides or polypeptides of the invention, includingfragments thereof.

Inhibition or reduction of the activity of S100 polynucleotides orpolypeptides may be useful in treating diseases, disorders, and/orconditions of the immune system, by inhibiting the proliferation,differentiation, or mobilization (chemotaxis) of immune cells. Theetiology of these immune diseases, disorders, and/or conditions may begenetic, somatic, such as cancer or some autoimmune diseases, disorders,and/or conditions, acquired (e.g., by chemotherapy or toxins), orinfectious. Moreover, inhibitors or antagonists of S100 polynucleotidesor polypeptides can be used as a marker or detector of a particularimmune system disease or disorder.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed by inhibitors of S100polynucleotides or polypeptides, or antagonists of S100 polynucleotidesor polypeptides. Moreover, these molecules can be used to treatanaphylaxis, hypersensitivity to an antigenic molecule, or blood groupincompatibility.

S100 polynucleotides or polypeptides are chemotactic molecules thatattract or mobilize cells (e.g., monocytes, fibroblasts, neutrophils,T-cells, mast cells, eosinophils, epithelial and/or endothelial cells)to a particular site in the body, such as an inflammation site, aninfection site, or a site of hyperproliferation. The mobilized cells canthen fight off and/or heal the particular trauma or abnormality.

Inhibitors or antagonists of S100 polynucleotides or polypeptides areprovided to decrease chemotactic activity to any immunological cells.These inhibitors or antagonists of S100 chemotactic molecules can thenbe used to treat and/or prevent inflammation, infection,hyperproliferative diseases, disorders, and/or conditions, or any immunesystem disorder by decreasing the number of cells targeted to aparticular location in the body. For example, inhibitors or antagonistsof S100 chemotactic molecules can be used to treat and/or prevent woundsinflammation and other trauma to tissues by neutralizing the attractionof immune cells to the injured location

Inhibition of S100 proteins can be achieved by using antibodies orinhibitors that bind or block access to the S100 proteins to a bindingsite or to any activation site activated by them.

The inhibitors or antagonists of S100 proteins can be employed toinhibit chemotaxis and activation of macrophages and their precursors,and of neutrophils, basophiles, B lymphocytes and some T cell subsets,e.g., activated and CD8+ cytotoxic T cells and natural killer cells, inauto-immune and chronic inflammatory and infective diseases. Examples ofauto-immune diseases include rheumatoid arthritis, multiple sclerosis,and insulin-dependent diabetes. Some infectious diseases includesilicosis, sarcoidosis, idiopathic pulmonary fibrosis caused bypreventing the recruitment and activation of mononuclear phagocytes,idiopathic hyper-eosinophilic syndrome caused by preventing eosinophilproduction and migration, endotoxic shock caused by preventing themigration of macrophages and their production of the chemokinepolypeptides of the present invention. The antagonists may also be usedfor treating atherosclerosis, by preventing monocyte infiltration in theartery wall.

The inhibitors or antagonists may also be used to treathistamine-mediated allergic reactions by inhibiting S100 protein-inducedmast cell and basophil degranulation and release of histamine.

The inhibitors or antagonists may also be used to treat inflammation bypreventing the attraction of monocytes to a wound area. They may also beused to regulate normal pulmonary macrophage populations, since acuteand chronic inflammatory pulmonary diseases are associated withsequestration of mononuclear phagocytes in the lung.

The inhibitors or antagonists may also be used to treat rheumatoidarthritis by preventing the attraction of monocytes into synovial fluidin the joints of patients. Neutrophil and monocyte influx and activationplay a significant role in the pathogenesis of both degenerative andinflammatory arthropathies.

The inhibitors or antagonists may be used to interfere with thedeleterious cascades attributed primarily to IL-1 and TNF, whichprevents the biosynthesis of other inflammatory cytokines. In this way,the antagonists may be used to prevent inflammation. The antagonists mayalso be used to inhibit prostaglandin-independent fever induced by S100chemokines.

Alternatively, the inhibitors or antagonists of S100 proteins can beused in conjunction with IL-10, which is involved in the down regulationof neutrophil migration at an inflamed site, such as for example, butwithout limiting it to, Crohn's disease or ulcerative colitis.

The inhibitors or antagonists of S00 proteins can also be used to treatcases of bone marrow failure, for example, aplastic anemia andmyelodysplastic syndrome. The inhibitors or antagonists may also be usedto threat cases of leukemia such as, but not restricted to acute myeloidleukemia, chronic myeloid leukemia, and acute lymphoid leukemia. Theinhibitors or antagonists can alternatively be used to treat or preventgraft rejection. The inhibitors or antagonists may also be used to treatasthma and allergy by preventing eosinophil accumulation in the lung.The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The S100 chemokine polynucleotides or polypeptides inhibitors andantagonists of the present invention may be employed in combination witha suitable pharmaceutical carrier. Such compositions comprise atherapeutically effective amount of the polypeptide, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The effects of S100 inhibitors or antagonists can be exploited inaccordance with the present invention through recombinant DNA expressionof these molecules, as well known in the art, of such inhibitors orantagonists in vivo, which is often referred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, the cells can be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering aninhibitor or antagonist of the present invention should be apparent tothose skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle, or can be alternatively any desirable expression vector orplasmid.

The inhibitors or antagonists of the present invention are targetedagainst S100 polynucleotides or polypeptides, which include, but are notlimited to, S100A8, S100A9, and S100A12, found as monomers, homodimersor heterodimers.

The inhibitors or antagonists can be an antibody used as a monoclonalantibody or a polyclonal antibody.

An antibody as defined herein, acting as inhibitor or antagonist of S100protein, can be administered alone or in combination with otherantibodies directed toward S100 polynucleotide or polypeptide.

The antibody is administered subcutaneously, intravenously,intramuscularly, intra-articular or intraperitoneally.

In one embodiment of the present invention, antibodies anti-S100proteins can be generated in a patient by simple immunization as it iswell known in the art. The immunization can be performed byadministration to a patient an S100 polypeptide or an S100 encodingpolynucleotide. The resulting immunization will allow to reduce orinhibit the chemotractant activity of the S100 proteins.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

EXAMPLE I Role Of S100A8 And S100A9 In Neutrophil Recruitment Induced ByMSU Crystals Material and Methods Recombinant Proteins and PolyclonalAntibodies

Murine S100A8 expression vector was a generous gift from Prof. H. J.Schluesener, (University of Tübingen, Germany). Murine S100A9 cDNA wassynthesized by RT-PCR from neutrophil RNA isolated using Trizol™ reagentaccording to the manufacturer's instructions (GibcoBRL, USA). S100A9cDNA was cloned into the pET28 expression vector (Novagen, Madison,Wis.) and transformed in E. coli HMS174. Recombinant protein expressionwas induced with 1 mM IPTG for 16 h at 16° C. After incubation, cultureswere centrifuged at 5,000×g for 10 min. The pellet was resuspended inPBS/NaCl 0.5 M/imidazole 1 mM and lysed by sonication. Lysates were thencentrifuged at 55,000×g for 25 min, supernatants collected and therecombinant His-tag S100A9 and S100A8 were purified using a nickelcolumn. His-tag proteins bound to the column were cleaved from theirHis-tag by adding 10 U of biotinylated thrombin and incubated for 16 hat room temperature. Recombinant S100A8 and S100A9 were eluted with PBS.The digestion and elution process was repeated once to cleave theremaining undigested recombinant proteins and biotinylated thrombin wasextracted from the eluates using streptavidin-agarose (Pierce, Rockford,Ill.). Contaminating LPS was removed on polymyxin B-agarose column(Pierce, Rockford, Ill.). LPS contamination was lower than 1 pg of LPSper μg of recombinant protein, as detected by the Limulus amoebocyteassay (Sigma, St-Louis, Mo.).

Polyclonal antisera against human and murine recombinant S100A8 andS100A9 were generated after repeated injections in New Zealand Whiterabbits or CD1 rats at 4 or 2 weeks intervals respectively. Antiseratiters were determined using direct ELISA and immunoblot. IgG fromantisera were purified by protein A affinity chromatography (PIERCE,Rockford, Ill.)

Air Pouch Experiments

Ten to twelve weeks old CD-1 or BALB/c mice were obtained from CharlesRiver, St-Colomban, Canada. Air pouches were raised on the dorsum bys.c. injection of 3 ml of sterile air on days 0 and 3. On day 7, 1.5 mgof MSU crystals suspended in a volume of 1 ml of endotoxin-free PBS(Sigma, St-Louis, Mo.) was injected into the air pouches. Alternatively,1 ml of murine S100A8 or S100A9 at concentrations ranging from 0.01 to10 μg/ml was injected into the air pouches. At specific times, the micewere killed by asphyxiation using CO₂, the air pouches were washed oncewith 1 ml of PBS-5 mM EDTA, and then twice with 2 ml of PBS-5 mM EDTA,and the exudates were centrifuged at 500×g for 5 minutes at roomtemperature. Cells were counted with a hematocytometer following aceticblue staining. Characterization of leukocyte subpopulations wasperformed by Wright-Giemsa staining of cytospin (VWR, Missisauga,Canada). In separate experiments, mice were injected i.p. 16 hours priorto injection of MSU crystals in the air pouch with 2 mg of purified IgGfrom rabbit antisera against murine S100A8 and S100A9 to inhibit theiractivities.

ELISAs

The detection of human and murine S100A8, S100A9, and S100A8/A9 wasperformed by coating 96-well plates with (100 μl/well) of humanS100A8/A9-specific mAb 5.5 (generous gift of Nancy Hogg, IORF, London,UK), purified rabbit IgG against mouse S100A8 or mouse S100A9 (for thedetection of murine S100A9 and S100A8/A9), diluted to a concentration of1 μg/ml in 0.1 M carbonate buffer pH 9.6. After overnight incubation,the plates were washed with PBS/0.1% Tween-20™ and blocked with PBS/0.1%Tween-20™/2% BSA for 30 min at room temperature. The samples andstandards (100 μl) were added and incubated for 1 hour at roomtemperature. After three washes with PBS/0.1% Tween-20™, the plates wereincubated for 1 hour at room temperature with 100 μl/well of 1/10,000dilutions of antisera against human S100A9 (for the detection of humanS100A8/A9) or with purified rat IgG against murine S100A9 or murineS100A8 (for the detection of murine S100A9, S100A8 and S100A8/A9). Theplates were then washed three times and incubated with 100 μl/well ofperoxidase-conjugated donkey anti-rabbit (1/7,500) (JacksonImmunoResearch, Mississauga, Canada) or peroxidase-conjugated Goatanti-rat (1/10,000) (Jackson ImmunoResearch, Mississauga, Canada) inPBS/0.1% Tween-20™/2% BSA for 1 hour at room temperature. After threewashes, the presence of IgG was detected with 100 μl of TMB-S accordingto the manufacturer's instructions and the OD was read at 500 nm.

Results

The activating potential of MSU crystals was first assessed to induce aninflammatory reaction in the murine air pouch model. As shown in FIG. 1,MSU crystals stimulated an important inflammatory reaction when injectedin the air pouch. Leukocyte recruitment was first detected 3 hours afterinjection and reached maximum levels within 9 hours, before returning tocontrol levels by 24 hours post-injection. More than 90% of therecruited leukocytes were neutrophils, the rest being monocytes.

Release Of MRPs In The Air Pouch In Response To MSU Crystals Injection

Knowing that high levels of MRPs are present in several inflammatoryprocesses, we therefore quantified the presence of MRPs in the air pouchexudates following MSU crystals injection. Low levels of S100A8, S100A9,and S100A8/A9 were detected in air pouch exudates of non-injected mice.Injection of MSU crystals led to the release of 7.5 μg/ml of S100A8/A9,which is approximately 1000 times more than chemokines. This release wasdetected as early as 3 hours post-injection and was maximal between 6 to12 hours following injection of MSU crystals. S100A8 and S100A9homodimers were also present but at inferior concentrations (FIG. 2A, 2Band 2C). The presence of MRPs in the pouch also correlated withneutrophil recruitment. These results suggested that MRPs could play arole in neutrophil recruitment in response to MSU crystals.

Role Of S100A8 And S100A9 In Neutrophil Recruitment Induced By MSUCrystals

To determine the role of MRPs in MSU-induced leukocyte recruitment, 10μg of recombinant murine S100A8 and S100A9 were first injected in theair pouch to determine their proinflammatory activities in vivo.Injection of both murine S100A8 and S100A9 led to the accumulation ofneutrophils in the air pouch. (FIG. 3A). Neutrophils recruitmentoccurred within 3 hours post-injection and was maximal between 6 and 9hours post-injection, after which time it returned to control levelswithin 24 hours (FIG. 3A). More than 95% of the migrated leukocytes wereneutrophils, with 5% of monocytes migrating as well. As shown in FIG.3B, S100A8, S100A9, and also S100A8/A9 induced leukocyte recruitment tothe air pouch in a dose-dependent fashion manner. Neutrophil recruitmentoccurred at injected doses as low as 0.1 μg, and was maximal at 10 μg.Those doses are similar to the levels detected in the air pouchesfollowing injection of MSU crystals (FIGS. 2A, B, and C).

The role of S100A8 and S100A9 in neutrophil migration induced by MSUcrystals was next investigated by inhibiting their activities usingpurified IgG from immunized rabbits. In preliminary experiments,anti-S100A8 and anti-S100A9 IgG specifically inhibited the recruitmentinduced in the air pouch following the injection of S100A8 and S100A9respectively. Peritoneal injection of purified IgG from pre-immunizedrabbits prior to MSU crystals injection in the air pouch slightlyreduces neutrophil recruitment (FIG. 4). Injection of anti-S100A8 alonereduced neutrophil recruitment by more than 50% (p<0.05, Dunnettmultiple comparison test). Moreover, injection of both anti-S100A8 andanti-S100A9 completely inhibited the neutrophil recruitment induced byMSU crystals to the air pouch (p<0.01). Since these antibodies bind toboth homodimers and S100A8/A9 heterodimers, injection of both antibodiescould have inactivated not only S100A8 and S100A9, but also S100A8/A9activity.

S100A8/A9 is Present in Synovial Fluids and Plasma of Patients SufferingFrom Gout

Inhibition By Anti-S100A8 And Anti-S100A9 Indicated That S100A8 andS100A9 were essential for neutrophil recruitment in MSU crystals-inducedinflammatory reaction in vivo. To verify whether they could play a rolein gout pathogenesis, we quantified S100A8/A9 by specific ELISA insynovial fluids and serum of several gout patients. S100A8/A9 was almostabsent from synovial fluids of osteoarthritis patients, a disease withno synovial inflammation (FIG. 5A). In contrast, up to 100 μg/ml weremeasured in synovial fluids of gout patients. S100A8/A9 was alsodetected in the serum of the same patients where it reached 1 μg/ml, aconcentration 100 times higher than measured in healthy donors (FIG.5B). These concentrations, which are higher than the ones detected inthe murine air pouch following MSU crystal injection, are consistentwith a role for S100A8 and S100A9 in gout pathogenesis.

Conclusion

The proinflammatory proteins S100A8 and S100A9 which are also present inthe air pouch exudates were found to induce neutrophil migration to theair pouch with a kinetic similar to MSU crystals. In addition,inactivation of both S100A8 and S100A9 led to a total inhibition ofneutrophil accumulation in response to MSU crystals, clearlydemonstrating their involvement in neutrophil recruitment in vivo. Sincethese proteins are also present at high concentrations in synovialfluids of gout patients, it is clear that they play an essential role ingout pathogenesis.

S100A8, S100A9, and S100A8/A9 were detected at high concentrations inthe exudates of mice injected with MSU crystals and in the synovialfluid of patients suffering from gout. The release was rapid, reaching10⁻⁸ M before 3 hours and close to 10⁻⁶ M within 6 hours post-injection.It was demonstrated that S100A8 and S100A9 are chemotactic atconcentrations of 10⁻¹⁰ to 10⁻⁸ M and stimulate neutrophil adhesion at10⁻⁷ to 10⁻⁶ M. This illustrates that they can direct neutrophilchemotaxis at early time points, before inducing their retention at theinflammatory site by stimulating their adhesion at later time. S100A8and S100A9 release also correlated with neutrophil recruitment in theair pouch exudate. Release of MRPs by neutrophils, and monocytes hasbeen demonstrated. The correlation between the release of MRPs andneutrophil recruitment, and the fact that 30% of the neutrophilcytosolic proteins are MRPs, shows that neutrophils are the primarysource of MRPs in the air pouch following MSU crystals injection.

Neutralization of S100A9 by passive immunization can reduce theneutrophil recruitment. Inactivation of S100A8 reduced neutrophilrecruitment by at least 50%. Passive immunization with anti-S100A8 andanti-S100A9 prior to injection of MSU crystals led to a total inhibitionof neutrophil recruitment to the air pouch suggesting that both S100A8and S100A9 play essential roles in the recruitment of neutrophils.Alternatively, injection of both antibodies could also inactivate theheterocomplex S100A8/A9, which is the major form found in the air pouchfollowing MSU crystals injection. As S100A8/A9 is also chemotactic forneutrophils and induces neutrophil accumulation in vivo, these resultssupport that S100A8/A9 can be exploited to play a role in MSUcrystals-induced recruitment.

It was also demonstrated that human S100A8, S100A9 and S100A8/A9 arechemotactic for neutrophil at concentration of 10⁻¹⁰ M in vitro. S100A9and S100A8/A9 are also shown to enhance monocyte adhesion and migrationacross endothelial cells via Mac-1/ICAM-1 interaction. Evidences werepresented here for the first time that S100A8 and S100A9 play achemotactic role in neutrophil migration in a mammal. The extracellularpresence of S100A8/A9 can therefore be associated to several pathologiesincluding rheumatoid arthritis, tuberculosis, ulcerative colitis, andCrohn's disease. This demonstrates that S100A8 and S100A9 play a role inother pathologies as well. This is also supported by the fact thatS100A12 (the third member of the MRP subfamily of S100 proteins) isinvolved in inflammation associated with experimentally-induced colitisand delayed-type hypersensitivity.

The present invention also contemplates a variety of usefulcompositions. For example, a preferred composition capable of inhibitinginflammation in animals comprises different S100 protein inhibitors,wherein said inhibitors are capable of inhibiting different inflammatoryreactions, as for example without limiting to, neutrophil migration, orsuperoxide production in phagocytic cells, in a pharmaceuticallyacceptable carrier or excipient. In a preferred embodiment, the animalis a human. Alternatively, preferred compositions according to thepresent invention may include any of the S100 protein inhibitordescribed hereinabove, for example, and without limitation, antibody,anti-sens mRNA, and antibody anti-chemokine factor receptor, to name buta few.

Another aspect of the invention relates to a method for directlyinhibiting activation of the specific inflammatory reaction byphagocytic cells, and more preferably, human phagocytic cells. A furtheraspect relates to methods for preventing or decreasing the tissue damageassociated with inflammatory reaction which involves administration oftherapeutically effective amount of S100 protein inhibitor as describedherein. The invention relates specifically to a method of preventing ordecreasing symptoms such as gout, autoimmune disorders, myocardialinfarction, adult respiratory distress syndrome (ARDS), asthma, andvarious dermatological disorders, which comprises the administration ofan effective amount of a S100 protein inhibitor or a derivative to apatient in need of such treatment.

The present invention also contemplates medicaments, and methods ofmaking same, many of which methods are well known in pharmaceuticalpractice. For example, the S100 protein inhibitors and derivatives ofthe present invention can be formulated into various forms foradministration to mucous membranes, into intra-articular areas,intraperitoneally, intravascularly, topically, subcutaneously, and viasuppository. Such medicaments may be formulated together with suitablecarriers, excipients, binders, fillers, and the like into dosage forms,with each form comprising a fraction or a multiple of the daily doserequired in order to achieve the desired treatment result.

It will also be appreciated that various combinations of the precedingelements may be made to provide other efficacious peptides,compositions, and methods according to the present invention.

EXAMPLE II Blockade Of S100 Proteins Suppresses Neutrophil Micgation InResponse To LPS Material and Methods Recombinant Proteins

Murine S100A8 cDNA cloned into the pET28a expression vector (Novagen,Madison, Wis.) was a generous gift from Professor Hermann J.Schlüesener, U. of Tubingen, Germany. Murine S100A9 cDNA was obtained byRT-PCR and cloned in our laboratory into the same vector. Recombinantproteins were produced as previously described (Ryckman et al., 2003, J.Immunol. 160: 1427). Contamination by endotoxins was lower than 1 pg/μgof recombinant proteins as assessed using the Limulus amoebocyte assay.Recombinant S100A8/A9 was produced by mixing together equimolarquantities of recombinant S100A8 and S100A9 in the presence of HBSSsupplemented with 10 mM HEPES, pH 7.4 containing 1.3 mM Ca²⁺.

Production of Polyclonal Antibodies

New Zealand White rabbits (<2.5 kg) were immunized by intradermal dorsalinjections (4 sites) with a total of 100 μg of purified murinerecombinant S100A8 or S100A9 in 500 μl endotoxin-free PBS (Sigma,St-Louis, USA) mixed with an equal volume of Freund's complete adjuvant.Antibody responses were enhanced by repeated injections 3 and 6 weeksafter the initial injection using the Freund's incomplete adjuvant.Antisera were collected and tested for specificity by ELISA and Westernblots against purified recombinant S100A8 and S100A9. Immunoglobulin G(IgG) from antisera was purified by protein A affinity chromatography(PIERCE, Rockford, Ill.). The anti-S100A8 antiserum had titers of1:100,000 and 1:500 for the detection in ELISA of 100 ng of S100A8 andS100A9 respectively. The anti-S100A9 antiserum had titers of 1:250 and1:100,000 for the detection in ELISA of 100 ng of S100A8 and S100A9respectively. Absence of cross reactivity of the purified IgG with theother murine myeloid related protein or proteins within the air pouchexudates was confirmed by immunoprecipitation assays and western blots.

CD Rats were immunized by i.p. injections with a total of 60 μg ofpurified murine recombinant S100A8 or S100A9 in 250 μl endotoxin-freePBS (Sigma, St-Louis, USA) mixed with an equal volume of Freund'scomplete adjuvant. Antibody response was enhanced by repeated injections14, 28, and 42 days after the initial injection using the Freund'sincomplete adjuvant. Antisera were collected and tested for specificityby ELISA and immunoblots against purified recombinant S100A8 and S100A9.The anti-S100A8 antiserum had titers of 1:10,000 and 1:500 for thedetection of 100 ng of S100A8 and S100A9 respectively. The anti-S100A9had titers of 1:250 and 1:10,000 for the detection of 100 ng of S100A8and S100A9 respectively.

ELISA

For S100A8 and S100A9, Costar High Binding 96-well plates (Corning,N.Y., USA) were coated overnight at 4° C. with 100 μl/well of purifiedrabbit IgG against S100A8 or S100A9 diluted to a concentration of 1μg/ml in 0.1 M carbonate buffer pH 9.6. The wells were blocked withPBS/0.1% Tween-20™/2% BSA (150 μl/well) for 30 min at room temperature.The samples and standards (100 μl) were added and incubated for 45 minat room temperature. The plates were washed 3 times with PBS/0.1%Tween-20™, and were incubated with rat IgG (100 μμl/well) against S100A8or S100A9 diluted in PBS/0.1% Tween-20™/2% BSA (1:10000) for 45 min atroom temperature. The plates were then washed 3 times in PBS/0.1%Tween-20™. To reveal the immune complex, peroxidase-conjugated goatanti-rat IgG (H+L) (minimum cross-reaction to rabbit serum proteins)(100 μl/well) at a dilution of 1:10000 was added and incubated 45 min atroom temperature. The plates were washed 3 times and 100 μl/well ofTMB-S substrate were added according to the manufacturer's instructions.The optical densities (ODs) were read at 500 nm. The lower limit ofquantification was determined as 4 ng/ml for both S100A8 and S100A9.

For S100A8/A9, 96-well plates were coated overnight at 4° C. withpurified anti-S100A9 rabbit IgG (μl/100 well) diluted 1 μg/ml in 0.1 Mcarbonate buffer pH 9.6. The wells were blocked with PBS/0.1%Tween-20™/2% BSA (150 μl/well) for 30 min at room temperature. Thesamples and standards (100 μl) were added and incubated for 45 min atroom temperature. The plates were washed 3 times with PBS/0.1% Tween-20™then incubated with 100 μl/well anti-S100A8 rat IgG diluted in PBS/0.1%Tween-20™/2% BSA (1:10000) for 45 min at room temperature. The plateswere next washed 3 times in PBS/0.1% Tween-20™ and incubated with 100μl/well of peroxidase-conjugated goat anti-rat IgG at a dilution of1:10000 for 45 min at room temperature. After 3 washes, 100 μl/well ofTMB-S substrate were added according to the manufacturer's instructions.The ODs were read on a plate reader at 500 nm. The lower limit ofquantification of this assay was determined as 10 ng/ml. All 3 ELISAswere tested using excess amounts (100 times) of S100A8, S100A9, orS100A8/A9 proteins and were shown to be specific under the conditionsreported here.

Air Pouch Experiments

The experimental protocols were approved by the Laval University animalprotection committee. Air pouches were raised on the dorsum of 10 to 12weeks-old CD-1 mice (Charles River, St-Colomban, Canada) by s.c.injection of 3 ml of sterile air on days 0 and 3 (Tessier et al., 1997J. Immunol. 159:3595). On day 7, 1 ml of LPS (1 μg/ml) or its diluent(PBS) was injected into the air pouches. At specific times, the micewere killed by asphyxiation using CO₂; peripheral blood was collected bycardiac puncture and diluted 1:20 in PBS-EDTA 5 mM. Total leukocyteswere stained with acetic blue and counted using a hematocytometer. Theair pouches were washed once with 1 ml PBS-5 mM EDTA, and then twicewith 2 ml of PBS-5 mM EDTA, and the exudates were centrifuged at 500×gfor 5 min at room temperature. Cells were counted with a hematocytometerfollowing acetic blue staining. Characterization of leukocytesubpopulations in the blood and migrating into the pouch space wasperformed by Wright-Giemsa staining of cytospins. In some experiments,mice were injected i.p. with 2 mg of purified rabbit IgG from preimmuneserum, anti-S100A8, or anti-S100A9 16 h before LPS injection in the airpouch.

Intravenous Injections

Animals were put on a heated cushion to dilate the tail vein 15 minbefore injection. Two hundred 1 of S100A8, S100A9, or S100A8/A9(0.006-60 μg/ml) was then injected i.v. in the tail vein of the mouse,corresponding to 0.05 to 500 μg of protein per kg of body weight.Animals were sacrificed by CO₂ asphyxiation at times ranging from 5 minto 24 h later; peripheral blood was collected by cardiac puncture anddiluted 1:20 in PBS-EDTA 5 mM. Total leukocytes were counted using ahematocytometer following acetic blue staining. Bone marrow cells werecollected by flushing with PBS-EDTA 5 mM through incisions made in thefemur, followed by disaggregation. Cytospin preparations of both bloodand bone marrow cells were analyzed after Wright-Giemsa differentialstaining.

Statistical Analyses

All statistical analyses were performed using the GraphPad Instat™software (GraphPad Software Inc., San Diego, Calif.). Statisticalcomparisons were made by analysis of variance (ANOVA) for the number ofleukocytes in air pouches, blood and bone marrow. The Bonferroni andDunnett multiple comparison tests were used to compare specific groupsat a confidence interval of 95%.

Results Release Of S100A8, S100A9, And S100A8/A9 In The ExtracellularMilieu Following Injection Of LPS

To examine the involvement of S100A8, S100A9, and S100A8/A9 inneutrophil migration, we first studied their release in vivo in responseto LPS. The air pouch model was selected since this closed environmentallows a clear measurement of immigrated leukocytes and releasedpro-inflammatory factors in the exudates. Few leukocytes were present inthe pouch exudates prior to the injection of either PBS or LPS.Injection of PBS in the air pouch led to a very mild accumulation ofneutrophils, probably consecutive to the injury caused by the needle. Incontrast, injection of LPS led to an inflammatory reaction associatedwith redness of the air pouch and the presence of plasma proteins in theair pouch exudates. Injection of LPS also induced the rapid migration ofleukocytes to the pouch, first detected 3h post-injection (FIG. 6A).This accumulation was maximal at 6 h post-injection and almost returnedto control levels by 12 h. More than 90% of the migrating leukocyteswere neutrophils, with few monocytes migrating as well.

This accumulation was associated with the release of S100A8, S100A9, andS100A8/A9 in the pouch exudates. Low levels of S100A8, S100A9, andS100A8/A9 were detected in the exudates of non-injected or PBS-injectedmice (FIGS. 6B-D). In contrast, injection of LPS led to the rapidrelease of all three S100 proteins. S100A8 was detected as early as 1 hpost-injection of LPS (before neutrophil migration, FIG. 6A) andremained significantly above the control levels for the next 23 h.Similarly, the presence of S100A9 was maximal between 3 and 12 hpost-injection of LPS, but the levels returned to control values by 24 hpost-injection. In contrast, the presence of S100A8/A9 was moretransitory, being maximal at 6 h post-injection of LPS and returning tocontrol levels by 9 h post-injection. While S100A9 and S100A8/A9concentrations were similar (3-5 μg/ml), S100A8 concentration was lower,reaching only 180 ng/ml. These results suggest that S100A8, S100A9, andS100A8/A9 are released separately during an inflammatory episode andprecede neutrophil immigration.

S100A8 And S100A9 Are Involved In Neutrophil Accumulation In Response ToLPS

To evaluate the role played by S100A8 and S100A9 in neutrophilmigration, mice were injected i.p. with purified rabbit IgG againstS100A8 and S100A9. LPS was then injected in the air pouches andneutrophil accumulation was measured 3 and 6 h later. Anti-S100A8 andanti-S100A9 had no effect on neutrophil accumulation in PBS-injectedmice (FIGS. 7A and B). Anti-S100A9 slightly reduced neutrophilaccumulation 3 h following injection of LPS, but this reduction was notsignificant (FIG. 7A). In contrast, anti-S100A8 reduced LPS-inducedneutrophil accumulation by 52% at 3 h post-injection (p<0.05, Bonferronitest). This inhibition was not enhanced by the addition of anti-S100A9.By 6 h post-injection, only the combination of anti-S100A8 andanti-S100A9 proved effective in preventing the migration of neutrophilsto the air pouch in response to LPS (FIG. 7B, p<0.05, Bonferroni test).These antibodies inhibited neutrophil migration by 82%.

Presence Of S100A9 and S100A8/A9 In The Serum Following Injection Of LPSIn The Air Pouch

LPS induced the accumulation of more than 5.4×10⁶ cells in the airpouches (FIG. 6A), twice the estimated number of neutrophil content ofthe blood (approximately 3×10⁶ cells). LPS therefore stimulated themigration of neutrophils to the air pouch in numbers greater than werepresent in the blood. This suggest that in mice LPS can either directlyor indirectly induce neutrophilia. To confirm this, LPS was injected inthe air pouches of mice and the number of neutrophils was evaluated inthe blood. Injection of LPS in the air pouch led to a 4.3 fold increasein the number of neutrophils circulating in the blood 3 h afterinjection (FIG. 8). This augmentation was transient, returning tocontrol levels at 6 h post-injection. The LPS-induced neutrophilia wasassociated with an increase in S100A9 and S100A8/A9 serum levels (FIGS.9A and B). Similarly to the number of circulating neutrophils, thisincrease was maximal at 3 h post-injection and almost returned tocontrol levels by 6 h post-injection, reaching a value of 292.9±66.0ng/ml of S100A9 and 595.3±172.0 ng/ml of S100A8/A9 3 h post-injection.Contrarily to S100A9 and S100A8/A9 levels, the concentrations of S100A8remained stable following injection of LPS in the air pouch.

Intravenous Injection Of S100A8, S100A9, And S100A8/A9 Results InNeutrophilia In Mice

The fact that the levels of S100A9 and S100A8/A9 correlated with theLPS-induced neutrophilia suggested that these proteins could participatein the neutrophilia associated with injection of LPS. The anti-S100A8and anti-S100A9 could therefore inhibit neutrophil migration indirectly,following a reduction of the circulating neutrophil caused by aninhibition of LPS-induced neutrophilia. To test this possibility,increasing doses of S100A8, S100A9, and S100A8/A9 were injected i.v. inmice and the peripheral blood was collected 3 hours later. As shown inFIGS. 10A, B and C, i.v. injection of S100A8, S100A9, and S100A8/A9caused an increase in the number of circulating neutrophils. The numberof neutrophils after injection reached 6.5, 2.7 and 7.4×10⁶ cells/ml inS100A8, S100A9, and S100A8/A9 injected mice respectively, compared toless than 1.5×10⁶ cells/ml for the control animals. This increase,detected for injected doses ranging from 5 to 500 μg/kg (0.12 to 12μg/mice), was significantly different from control (p<0.05, Dunnettmultiple comparison test) and maximum at a dose of 50 to 250 μg/kg.Although the total number of circulating leukocytes increased slightlyin S100 protein-injected mice, this increase was not significantlydifferent from that in PBS-injected mice. Assuming a total blood contentof 79 ml/kg, these doses corresponded to serum concentrations rangingapproximately from 600 to 3000 ng/ml at the time of injection. Thesedoses are similar to the ones measured following injection of LPS in theair pouch (FIGS. 6 B-D).

S100A8, S100A9, And S100A8/A9 Induce The Release Of Bone MarrowNeutrophils

The kinetic study of S100A8 and S100A9 injection over a 24 h period(FIGS. 11A and B) showed that they induced neutrophilia over a period of3 to 6 h post-injection. At 3 h, the number of neutrophils was2.8×10⁶±0.5×10⁶ cells/ml in S100A8-injected mice and 3.5×10⁶±0.7×10⁶ inS100A9-injected mice, compared to 1.0×10⁶±0.2×10⁶ cells/ml for thecontrol mice (p<0.05, Bonferroni test). The increase in circulatingneutrophils returned to the control levels by 12 h post-injection. Theincrease in the number of neutrophils in the blood induced by S100A8 andS100A9 closely correlated with a decrease in those of the bone marrow(FIGS. 11C and D). Approximately 22 to 27% of the bone marrow cells innon-injected mice were segmented and non-segmented neutrophils. Thispercentage did not vary significantly in PBS-injected mice. In contrast,the proportion of neutrophils decreased by 50% in bone marrow cells 3and 6 h post injection of S100A8 or S100A9 (p<0.01 and p<0.05,respectively). This strongly suggest that S100A8 and S100A9 induce therelease of neutrophils from the bone marrow to the blood.

Anti-S100A8 And Anti-S100A9 Inhibit The Neutrophilia Induced ByInjection Of LPS In The Air Pouch

To evaluate the role played by S100A8 and S100A9 in LPS-inducedneutrophilia, mice were injected i.p. with purified rabbit IgG againstS100A8 and S100A9. LPS was then injected in the air pouches and thenumber of circulating neutrophils was measured 3 h later. As shown inFIG. 12, injection of anti-S100A9 led to an almost complete inhibitionof the neutrophilia associated with the local injection of LPS (p<0.05Bonferroni test). This inhibition was not increased when anti-S100A8 andanti-S100A9 were injected together. Although the anti-S100A8 alsodiminished the neutrophilia associated with LPS injection, thisinhibition was not significant. As expected, the anti-S100A8,anti-S100A9, and the control IgG had no effect on the number ofcirculating neutrophils in PBS-injected mice.

Discussion

In the present experiment, it was demonstrated that S100A8, S100A9, andS100A8/A9 are released in the air pouch exudates and serum during aninflammatory reaction induced by LPS. Their presence in the exudatespreceded the migration of neutrophil to the air pouch, while S100A9 andS100A8/A9 presence in serum correlated with LPS-induced neutrophilia.S100A8, S100A9, and S100A8/A9 induced the release of neutrophils fromthe bone marrow to the blood when injected i.v. and neutrophilaccumulation when injected in the air pouch. Finally, passiveimmunization with purified IgG against S100A8 and S100A9 resulted in theinhibition of neutrophilia and neutrophil migration to the air pouch.

The kinetic of neutrophil accumulation to the air pouch offers a way ofdeciphering the role of S100A8 and S100A9 in the sequential steps of themechanism of neutrophil migration from the bone marrow to theinflammatory site. Preliminary results using intravital microscopydemonstrated that neutrophil emigration from the blood vessel to the airpouch tissue begins within the first hour following injection of LPS andthat neutrophils do not reach the air pouch lumen before 2 hpost-injection. Consequently, exudates neutrophils at 3 h post-injectionemigrated from the blood at the most 1 h post-injection. As shown inFIG. 8, the number of circulating neutrophils is not increased at 1 hpost-injection. This indicates that exudates neutrophils at 3 hpost-injection originate mostly from the pre-injection peripheral bloodpool of neutrophils. Blocking molecules at the 3 h time point thereforeprovide indications about the role of the blocked molecule in neutrophilmigration from the blood to the inflammatory site. In contrast, by 6 hpost-injection, neutrophils had enough time to be released from the bonemarrow storage pool, circulate in the peripheral blood and emigrate tothe exudates. As a consequence, inhibition by blocking antibodies at 6 hpost-injection can be due to the inhibition of neutrophil release fromthe bone marrow or neutrophil migration to the inflammatory site. Thesetwo possibilities can be further resolved by analysing the effect of theblocking antibodies on the numbers of circulating blood neutrophils at 3h post-injection of LPS.

By analysing the effect of the blocking Abs at the two time points, itcan be concluded that S100A8 and S100A9 play a role at the levels ofboth neutrophil migration to the air pouch and neutrophil release fromthe bone marrow respectively. Proofs of this comes from the fact thatanti-S100A8 inhibited neutrophil migration to the air pouch at 3 hpost-injection of LPS (FIG. 7A), but failed to significantly reduceneutrophil release from the bone marrow (FIG. 12). In addition, by 3 hpost-injection, 2.1×10⁶ neutrophils had migrated to the air pouch, whichis less than the approximately 3×10⁶ neutrophils circulating in theblood of a resting mouse. This indicates that by 3 h post-injection, thepouch neutrophils originated from the circulating, but not the bonemarrow storage pool of neutrophils. Since no increase in peripheralblood neutrophils was detected in LPS-injected mice before 3 hpost-injection, this suggests that the anti-S100A8 IgG directlyinhibited neutrophil migration to the air pouch. Therefore, the role ofS100A8 would be to assist in neutrophil migration to the inflammatorysite. Support for this hypothesis comes from the fact that murine S100A8was found to be chemotactic for neutrophils, and to activate Mac-1, anintegrin important in neutrophil transendothelial migration.

At 6 h post-injection of LPS, the combination of anti-S100A8 andanti-S100A9 inhibited neutrophil migration to the air pouch (FIG. 7B).

It is contemplated that the polypeptides, compositions and methods ofthe present invention may also be useful in veterinary applications, aswell as in the treatment of humans.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A method for the treatment of an inflammatoryreaction in a human suffering therefrom comprising administering to saidhuman an inflammatory reaction treatment effective amount of an antibodyor a fragment thereof directed against S100A8 protein, S100A9 or acombination thereof.
 2. The method of claim 1, wherein said inflammatoryreaction is selected from the group consisting of arthritis, chronicpolyarthritis, rheumatoid arthritis, gout, asthma, psoriasis,paraneoplastic syndrome, tumor-induced inflammatory diseases, turbideffusions, collagenosis, postinfectious arthritis, seronegativespondylarthritis, vasculitis, sarcoidosis, arthrosis, Crohn's disease,ulcerative colitis, tuberculosis, cell chemotaxis, cell migration, cellrecruitement, proteolysis, oxidative burst, radical oxidation, acutemyeloid leukemia, chronic myeloid leukemia or acute lymphoid leukemiaand graft rejection.
 3. The method of claim 1, wherein said inflammatoryreaction is selected from the group consisting of rheumatoid arthritis,Crohn's disease, ulcerative colitis, and tuberculosis.
 4. The method ofclaim 1, wherein said inflammatory reaction is selected from the groupconsisting of rheumatoid arthritis and Crohn's disease.
 5. The method ofclaim 1, wherein said administration is performed by intravenous, oral,intranasal, subcutaneous, topical, or intraperitoneal administration.